We have recently resumed our work on platelet and blood clotting inhibition by nitrite and are studying the effects of changes in ambient oxygen levels on the processes in rodent models using the TEG methodologies. We have also measured the effects of red cell reduction of plasma nitrite on platelet signaling because of some confusion in the literature and have confirmed that VASP phosphorylation occurs with the generation of NO but that this signal is very dependent on the kinetics of reaction and the exact experimental conditions, especially red cell concentrations. The VASP phosphorylation appears to be an order of magnitude more sensitive than the direct measurements of cGMP that we have used in the past. These measurements have been very valuable for our collaborators in Bangkok, Thailand who have found such changes in circulating platelets in certain patients after sodium nitrite inhalation (see DK 025104). Because of the recent interest in the results of VASP phosphorylation studies we have published further on this process and produced a Journal of Visualized Research tutorial so others may better use this assay. Our studies of skeletal muscle nitrate, which showed that during exercise the nitrate could be reduced to nitrite and then NO, has focused on better understanding of both the reductive processes and how muscle obtains such high levels of nitrate. So far our results are still most compatible with xanthine oxido-reductase as the major enzyme involved in the reductive processes. Dietary and genetic manipulations of rodents has shown that NOS 1 (nNOS) and myoglobin knockouts have markedly reduced levels of skeletal muscle nitrate suggesting that, as we predicted, both are involved in these high levels. However, we also find that dietary limitations of nitrate and nitrite lower these levels greatly (more than in blood or liver) and that return of these ions to the diet results in rapid accumulation and, indeed, in some cases an overshoot of the levels. These results are also discussed in the accompanying report (DK 025104). This result raises the possibility of some active transport mechanisms in the muscle, perhaps by a protein similar to sialin which transports nitrate into the saliva from blood. We are also currently studying these processes in muscle cells, primary and continuous lines, in culture, including in cells which can be caused to differentiate from myoblasts to myocytes. Preliminary results suggested that differentiation of muscle cells is required for enhancing nitrate reduction to nitrite and allowed us to quantitate the roles of n-nitric oxide synthase, myoglobin, sialin, a chloride transporter protein, etc in affecting the levels of nitrate in muscle tissues. One paper on this work has been published and another is under revision after initial submission.